Genome sequences of human and many other organism have been analyzed, and efficient and logical genome-based drug discovery based on the obtained genomic information now draw attention. Most of the targets of the drugs are proteins. The number of proteins synthesized from human genes is estimated to be about 32,000 in total. Considering the post-translational modifications, the number of such proteins is several times more than the number simply calculated based on the number of human genes. Also considering there are drugs which do not directly act on the humans such as antibiotics, a huge number of proteins are to be analyzed for drug discovery. However, only a limited number of proteins can be actual targets of drugs.
Recently, once a specific target protein is determined for drug discovery, it has become possible to screen an optimum compound for the target among many compounds by a technique such as HTS (High Throughput Screening) or the like. This is now a main style for drug discovery studies. However, specificity (binding, interaction, etc.) of the selected compound has been merely confirmed with respect to several kinds of proteins. No comprehensive review on the specificity has been performed.
Identification of true target molecules of the drug candidates and information transmission paths involving the target molecules clarifies mechanism of drug efficacy and side effects, and thus plays an important role in clinical tests for differentiating the drug of interest from other drugs in efficacy, side effects or the like. There are several techniques for searching for target molecules of drugs, but biochemical experiments are required at a final stage anyway to directly prove the interaction between the drugs and target proteins.
Proteomics has advantages over genetics in that proteins binding to drugs can be directly analyzed with a mass spectrometer (MS) and that the protein samples may be derived from cultured cell or organs.
As a technique for isolating a specific proteins, affinity chromatography is widely used. As a representative example of using affinity chromatography for the purpose of isolating proteins bound to compounds, identification of cis-trans peptidyl-prolyl isomerase (FKBP) as a target protein of immunosuppressant drug FK506, was cited1,2). Harding et al. immobilized FK506 to an affinity matrix with the activity being retained, and flowed lysate prepared from spleen. They competitively eluted proteins bound to a ligand with a non-immobilized FK506 solution, and thus obtained FKBP as a specific binding protein. Some physiologically active natural substances strengthen activity thereof by covalently binding to the target3-5). A tag such as biotin is introduced into such a substance, and the resultant substance is added to a cell and incubated for a certain time period to prepare lysate. From the obtained lysate, a compound labeled with a biotin tag is collected using a column having avidin immobilized thereon. In this way, the protein bound to the compound can be identified. Sin et al. synthesized a biotinated derivative of fumagillin having an angiogenesis suppressive activity, and reacted the biotinated derivative with the bovine brain extract. After performing several stages of chromatography, they collected the biotinated derivative using an avidin column, and identified type II methionine aminopeptidase (MetAP-2) as a specifically binding protein. Unlike the affinity chromatography with the compound immobilized thereon, this system enable the reaction of a cultured cell, as well as in a cell extract, and a probe under physiological conditions. This is effective for isolating binding proteins which are easily denatured during cell fractionation or lysate preparation5).
Affinity chromatography may occasionally identify proteins not originally expected6-9). Knockaert et al.7) evaluated various cycline-dependent kinase (CDK) inhibitors, first with an in vitro kinase panel measurement system. The enzymes used were quite limited, mainly several types of CDK and glycogen synthase kinase-3α/β (GSK-3α/β). This assay demonstrated that paullone is an inhibitor against CDK and GSK-3α/β. When paullone immobilized to an affinity matrix was used as a probe for purifying binding proteins, an unexpected result was obtained. It was found that mitochondrial malate dehydrogenase (mMDH) specifically binds to this probe as well as these types of kinase. In an in vitro enzymatic activity measurement performed thereafter, it was confirmed that paullone inhibits mMDH activity with certainty. A technique using affinity chromatography clarified an unexpected novel intracellular target (mMDH) regarding paullone for the first time in history. This experimental result is considered to provide some suggestions to the studies on targets and action mechanisms of compounds. One of the suggestions is that the action of the compound may possibly be expressed as a total of a plurality of mechanisms, not only as the expected mechanism via the target protein.
Affinity chromatography is based on non-covalent interaction between compounds and proteins except for some physiologically active natural substances. However, when the affinity is weak, a true target may be dissociated from the compound during the operation. For example, a compound may be saturated with a protein having a stronger affinity or a protein having an equal strength of affinity but contained in a larger amount. Namely, proteins obtained by affinity chromatography include a certain amount of proteins bound by non-specific interaction. Especially, a drug having a high protein binding ratio can possibly be bound to a great number of proteins in serum or the like. Such a drug often has 200 to 300 kinds of proteins binding thereto even after, for example, being thoroughly washed with 1 M sodium chloride in an affinity column or the like. Therefore, even though the proteome technology has been developed to make it relatively easy to identify many proteins, how to find specific proteins from these identified proteins is important. Shimizu et al. coated a matrix surface with a highly hydrophilic polymer having a high molecular weight and thus reduced the non-specific adsorption of the matrix as compared to generally used matrices2).
Another attempt to reduce the non-specific interaction is to narrow a range of a group of proteins which may be specifically bound to the compounds before affinity chromatography is performed10). In order to examine proteins bound to chloroquine, which is an anti-malaria drug having a quinoline backbone, a group of ATP-binding proteins are first purified by a column having ATP (adenosine triphosphate) immobilized thereon to obtain a group of binding protein candidates. Quinoline, which has a structure similar to that of purine, is considered to have affinity to proteins bound to purine nucleotides such as ATP and DNA. Therefore, by narrowing a range of targets as target candidates using an ATP column in advance, the non-specific interaction was reduced and thus target proteins of chloroquine were identified. This clarified that chloroquine acts on humans, not on malaria, and thus the action mechanism and the side effect mechanism were estimated.
Another attempt to increase the specificity is to competitively elute binding proteins from a compound-immobilized column, using non-immobilized compounds, molecules which are considered to compete for binding sites (e.g., ATP or NADH (nicotinamide adenine dinucleotide)) or the like. An analysis of proteins bound to p38 inhibitor SB203580 successfully increased the p38 recovery ratio by eluting compounds together with ATP11). By this method, a great number of new types of kinase bound to SB203580, which had been considered to p38-specific. Such competitive elution of compounds requires that sufficient amount of the compounds should be dissolved in an aqueous solution, and it is difficult to apply this method to a poorly soluble drug. However, by mixing a non-immobilized, free compound to a protein mixture solution before applied to the affinity column, the target can be masked. A sample containing the masked protein is applied to the affinity column, and column-binding proteins are separated by SDS-PAGE. The bands on the SDS-PAGE are compared based on whether or not the protein is masked or not. The band which disappeared by being masked is considered to be the target2). Even a compound having a poor water solubility is expected to be dissolved to a certain degree in a protein mixture solution.
However, compounds having a low molecular weight are mostly bound to serum proteins to some extent. Therefore, target proteins specific to compounds having a low molecular weight need to be found from binding proteins.
This is realized by preparing a compound having a similar structure to that of, but a different activity from that of, the intended compound and performing differential display of binding proteins. SDS-PAGE may be performed to find different bands, but it is not easy to separate all the bands where there are many binding proteins. Under the circumstances, the present inventor reviewed a target protein identification technique for a novel anti-cancer drug E7070 by performing MS analysis12) using two-dimensional electophoresis or stable isotope labeling, and reported the results13). E7070 is a compound screened, using an indication that a cell cycle of a mouse derived cancer cell P388 is inhibited by G1 phase14). The binding proteins to E7070 had been unknown. The present inventor attempted to identify the binding proteins using E7070 as an affinity probe. With a technique using a probe, the quality of the probe is a key factor. It is ideal that the probe has about the same degree of activity as that of the original compound. A compound has a structure indispensable to express the activity thereof. The indispensable structure is clarified based on the structural activity relationship while a chemist converts the structure of the compound and improves the activity. A linker is extended from a part which is not the indispensable structural part to introduce the probe to the matrix. With E7070, a probe was synthesized by converting a sulfamoyl group based on the structural activity relationship. Binding proteins were identified by affinity chromatography having E7070 immobilized thereon. However, a huge number of proteins were bound to the probe, and it was difficult to narrow the range of the target proteins. In general, synthetic compounds having a low molecular weight have a low specificity and tend to bind to various proteins. Synthetic compounds having a low molecular weight such as E7070 are often poorly water-soluble, and therefore it is often difficult to specifically elute such compounds by flowing non-immobilized drugs to the affinity column in a huge amount. E7070 was also poor in water solubility, and had to be eluted using a solution such as a surfactant or a denaturing agent increasing the elution capability of the solution step by step. Various purification conditions were reviewed. With E7070, most of the eluted proteins were bound to the compound, and there was no non-specific adsorption to beads or linker. The present inventor newly prepared probes using compounds having a similar structure to, but a different anti-tumor activity from, E7070. The proteins eluted from each of the probes were analyzed using a quantitative proteome technique of ICAT15, 16) and 2D-DIGE17) to construct a strategy for identifying proteins strongly adsorbing to E707013). As a result, the present inventor successfully identified proteins highly specifically adsorbing to an E7070 type probe among a great number of proteins adsorbing to the two matrices. Unlike the conventional qualitative determination based on the presence/absence of the band by SDS-PAGE, such a quantitation can provide a highly reliable result.
However, the above-described method still has some problems as exemplified below. (1) Although the compounds need to be immobilized on the carrier (encompassing a carrier filling the column; hereinafter, occasionally referred to simply as “column”), most of the compounds synthesized for screening occasionally cannot be immobilized on the carrier (column). Therefore, there is a need to separately synthesize compounds, which requires a huge amount of time and labor. The type of binding proteins often vary in accordance with the site of the compound immobilized on the carrier (column). (2) When changing the structure of a compound such that the compound can be immobilized on the carrier (column), the activity should not be changed. However, the specificity or affinity is occasionally changed by changing the structure. (3) When performing differential display by changing the compound to be immobilized on the affinity column, it is difficult to select negative compounds (compounds having a relatively weak activity) rather than positive compounds (compounds having a relatively strong activity). Ideally, all the negative compounds are immobilized on the affinity column, but it is not easy in actuality for reason (1). (4) In general, the amount of compounds immobilized on the affinity column is often 0.1 mg to several milligrams with respect to 1 ml of gel, which corresponds to about 1 mM. However, a drug inducing some phenotype of a cell (having activity) shows activity by the order of several micromoles to several nanomoles, sometimes by the order of picomoles. The concentration of the drug exhibiting activity is significantly different from the concentration of the drug immobilized on the column. In general, when a drug is used at a significantly higher concentration than the dose of efficacy, non-specific toxicity is often exhibited. Namely, it is considered that when a high concentration drug is immobilized on the column, an area exhibiting such toxicity may be involved. It is possible to reduce the amount of the compounds immobilized on the column from several micromoles to several nanomoles. However, it is not necessarily easy to control such a tiny amount of immobilization, because if the amount of binding proteins, i.e., the load of the affinity column, is significantly reduced, there is a possibility that the intended proteins may become undetectable with MS. In order to avoid this, the column size may be increased 100 to 1000 times, but such a large size of column is difficult to handle for practical use.